Enhanced heat shock protein (HSP) expression protects cells and tissues from injury. Further, enhanced HSP expression improves survival in experimental models of critical illness. However, these findings have not been applied in a clinical setting, as laboratory inducers of HSPs are not safe for human administration. Thus, this powerful tool, that may significantly improve clinical outcome, has yet to be utilized. Our laboratory has shown glutamine (GLN) can safely enhance HSP expression in tissues of critically ill and injured animals and established HSP induction is necessary for GLN's beneficial effect following experimental illness. In a trial of critically ill patients we demonstrated GLN enhanced HSP-70 levels, which correlated with improved outcome. However, the mechanism by which GLN induces HSP expression is unknown. We hypothesize GLN induces HSP expression via activation of the O-linked glycosylation pathway (O-GlcNAc), which is known to depend on GLN as a rate limiting substrate. This pathway can activate key transcription factors required for HSP induction. Our preliminary data indicates GLN increases the activity of the O-GlcNAc pathway and nuclear translocation and activation of key inducers of the HSP pathway, such as Sp1 and heat shock factor-1 (HSF-1). Further, siRNA inhibition of one of the key O-GlcNAc pathway enzymes significantly blunts the GLN-mediated increase in HSP expression. The major focus of this proposal is to determine the mechanism by which GLN induces HSP expression. Our hypothesis is GLN acts via transport into the cell and metabolism by the O-GlcNAc pathway to increase O-linked glycosylation of key transcription factors required for HSP gene activation, which then increases the expression of HSPs. To address this hypothesis, we propose three specific aims: This project will utilize cellular and animal models of illness/injury and employ chemical/genetic inhibition of key enzymes in these pathways to address the following specific aims: 1) Evaluate the role of GLN transport and metabolism via the O-GlcNAc pathway in stress/injury. 2) Evaluate via promoter truncation which promoter regions are key for GLN-mediated HSP expression. Then, determine the effect of GLN on nuclear translocation and transactivation of key transcription factors responsible for HSP expression. 3) Evaluate effects of GLN transport, metabolism, and transcriptional activation on the following cellular and in vivo endpoints: a) HSP expression (multiple families of HSPs), b) tissue/cellular injury, and c) cell volume. This project will elucidate how GLN induces HSP expression in clinical illness and injury. We believe GLN will be able to be administered as a pharmacologic agent prior to surgery or at onset of critical illness/tissue injury (with admittance to ICU/emergency room) to enhance HSP expression and improve survival. This project will elucidate how glutamine induces protective heat shock protein expression in clinical illness and injury. We believe glutamine will be able to be administered as a pharmacologic agent prior to surgery or at onset of critical illness/tissue injury (with admittance to ICU/emergency room) to enhance heat shock protein expression and improve survival.